Negative cell-cycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells

Abstract

Haematopoietic stem cells (HSCs) are capable of shifting from a state of relative quiescence under homeostatic conditions to rapid proliferation under conditions of stress. The mechanisms that regulate the relative quiescence of stem cells and its association with self-renewal are unclear, as is the contribution of molecular regulators of the cell cycle to these decisions. Understanding the mechanisms that govern these transitions will provide important insights into cell-cycle regulation of HSCs and possible therapeutic approaches to expand HSCs. We have investigated the role of two negative regulators of the cell cycle, p27Kip1 and MAD1, in controlling this transition. Here we show that Mad1−/−p27Kip1−/− bone marrow has a 5.7-fold increase in the frequency of stem cells, and surprisingly, an expanded pool of quiescent HSCs. However, Mad1−/−p27Kip1−/− stem cells exhibit an enhanced proliferative response under conditions of stress, such as cytokine stimulation in vitro and regeneration of the haematopoietic system after ablation in vivo. Together these data demonstrate that the MYC-antagonist MAD1 and cyclin-dependent kinase inhibitor p27Kip1 cooperate to regulate the self-renewal and differentiation of HSCs in a context-dependent manner.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Expansion of the phenotypic stem cell population in Mad1−/− p27Kip1−/− bone marrow.
Figure 2: Increased numbers of long-term repopulating cells following loss of MAD1 and p27Kip1.
Figure 3: Increased quiescent stem cell pool in Mad1−/− and Mad1−/− p27Kip1−/− bone marrow under homeostatic conditions.
Figure 4: Loss of both MAD1 and p27Kip1 enhanced recovery of haemopoiesis in vivo under conditions of stress.

References

  1. 1

    Bradford, G. B., Williams, B., Rossi, R. & Bertoncello, I. Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment. Exp. Hematol. 25, 445–453 (1997).

  2. 2

    Lemischka, I. R., Raulet, D. H. & Mulligan, R. C. Developmental potential and dynamic behaviour of hematopoietic stem cells. Cell 45, 917–927 (1986).

  3. 3

    Jordan, C. T., Yamasaki, G. & Minamoto, D. High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations. Exp. Hematol. 24, 1347–1355 (1996).

  4. 4

    Leary, A. G., Zeng, H. Q., Clark, S. C. & Ogawa, M. Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors. Proc. Natl Acad. Sci. USA 89, 4013–4017 (1992).

  5. 5

    Li, C. L. & Johnson, G. R. Stem cell factor enhances the survival but not the self-renewal of murine hematopoietic long-term repopulating cells. Blood 84, 408–414 (1994).

  6. 6

    McArthur, G. A. et al. MAD1 and p27KIP1 cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity. Mol. Cell. Biol. 22, 3014–3023 (2002).

  7. 7

    Zezula, J. et al. p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal. EMBO Rep. 2, 27–34 (2001).

  8. 8

    Holzel, M. et al. Myc/Max/Mad regulate the frequency but not the duration of productive cell cycles. EMBO Rep. 2, 1125–1132 (2001).

  9. 9

    Fero, M. L. et al. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27Kip1-deficient mice. Cell 85, 733–744 (1996).

  10. 10

    Foley, K. P. et al. Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation. EMBO J. 17, 774–785 (1998).

  11. 11

    Till, J. & McCulloch, E. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213–222 (1961).

  12. 12

    Purton, L. E., Bernstein, I. D. & Collins, S. J. All-trans retinoic acid delays the differentiation of primitive hematopoietic precursors (lin-c-kit+Sca-1+) while enhancing the terminal maturation of committed granulocyte/monocyte progenitors. Blood 94, 483–495 (1999).

  13. 13

    Akashi, K., Traver, D., Miyamoto, T. & Weissman, I. L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404, 193–197 (2000).

  14. 14

    Kondo, M., Weissman, I. L. & Akashi, K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672 (1997).

  15. 15

    Okada, S. et al. In vivo and in vitro cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood 80, 3044–3050 (1992).

  16. 16

    Cheng, T., Rodrigues, N., Dombkowski, D., Stier, S. & Scadden, D. Stem cell repopulation efficiency but not pool size is governed by p27kip1. Nature Med. 6, 1235–1240 (2000).

  17. 17

    Osawa, M., Hanada, K., Hamada, H. & Nakauchi, H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273, 242–245 (1996).

  18. 18

    Szilvassy, S. J., Humphries, R. K., Lansdorp, P. M., Eaves, A. C. & Eaves, C. J. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc. Natl Acad. Sci. USA 87, 8736–8740 (1990).

  19. 19

    Taswell, C. Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J. Immunol. 126, 1614–1619 (1981).

  20. 20

    Sato, T., Laver, J. H. & Ogawa, M. Reversible expression of CD34 by murine hematopoietic cells. Blood 94, 2548–2554 (1999).

  21. 21

    Tajima, F., Sato, T., Laver, J. H. & Ogawa, M. CD34 expression by murine hematopoietic stem cells mobilized by granulocyte colony-stimulating factor. Blood 96, 1989–1993 (2000).

  22. 22

    Cheng, T. et al. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 287, 1804–1808 (2000).

  23. 23

    Gothot, A., Pyatt, R., McMahel, J., Rice, S. & Srour, E. F. Functional heterogeneity of human CD34+ cells isolated in subcompartments of the G0/G1 phase of the cell cycle. Blood 90, 4384–4393 (1997).

  24. 24

    Spangrude, G. J. & Scollay, R. A simplified method for enrichment of mouse hematopoietic stem cells. Exp. Hematol. 18, 920–926 (1990).

  25. 25

    Gothot, A., van der Loo, J. C., Clapp, D. W. & Srour, E. F. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice. Blood 92, 2641–2649 (1998).

  26. 26

    Lerner, C. & Harrison, D. E. 5-Fluorouracil spares hemopoietic stem cells responsible for long-term repopulation. Exp. Hematol. 18, 114–118 (1990).

  27. 27

    Hodgson, G. S., Bradley, T. R. & Radley, J. M. The organisation of the hemopoietic tissue as inferred from the effects of 5-fluorouracil. Exp. Hematol. 10, 26–35 (1982).

  28. 28

    Walkley, C. R. et al. Identification of the molecular requirements for an RARα-mediated cell cycle arrest during granulocytic differentiation. Blood 103, 1286–1295 (2004).

  29. 29

    Harrison, D. E., Jordan, C. T., Zhong, R. K. & Astle, C. M. Primitive hemopoietic stem cells: direct assay of most productive populations by competitive repopulation with simple binomial, correlation and covariance calculations. Exp. Hematol. 21, 206–219 (1993).

  30. 30

    Spangrude, G. J., Heimfeld, S. & Weissman, I. L. Purification and characterization of mouse hematopoietic stem cells. Science 241, 58–62 (1988).

Download references

Acknowledgements

We thank E. Sitnicka and C. Li for discussion and critical comment, Peter MacCallum Cancer Centre Animal Facility Staff for care of experimental animals, FACS staff for assistance with FACS sorting. C.R.W. is a recipient of an Australian Postgraduate Award. This work was supported by grants from the National Health & Medical Research Council of Australia (NHMRC) to G.A.M and L.E.P.

Author information

Correspondence to Louise E. Purton or Grant A. McArthur.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Fig S1, Fig S2, Fig S3, Table 1 (PDF 97 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading